The immediate objectives of this proposal are to shed light on the role of Tat and P-TEFb in HIV-1 latency and reactivation, and to investigate the regulation of these proteins by the cellular factors HIC and granulin. P-TEFb (cyclin T1/CDK9) is a cellular transcription elongation factor that cooperates with the viral protein Tal to attain processive transcription of the HIV genome. We will study the control of HIV infection by naturally occurring variations in Tat sequences derived from HIV patients' memory T cells, and the effects on P-TEFb and virus production that accompany the activation of latently infected cells. The novel interactions of HIC and granulin with Tat and cyclin T1 will be characterized to understand how they modulate HIV gene expression. Long-term implications of these investigations lie in the control of HIV reservoirs and the development of compounds to interfere with HIV-1 infection. Our specific aims are to: 1. Test the hypothesis that partially active Tat mutants are associated with viral latency. Work with latently infected cell lines and clinical samples suggests that impaired Tat transactivation activity may contribute to the establishment of latency. Our studies have shown that the transactivation activity of moderately debilitated Tat mutants is preferentially stimulated by a mitogen that induces P-TEFb. To explore the role of Tat attenuation in latency, we will (i) examine the Tat sequences present in viruses that emerge after activation of resting CD4+ T cells from patients on HAART, (ii) study cells infected with viruses containing partially active Tat variants, and (iii) characterize the variants biochemically. 2. Explore the relationship between P-TEFb activity and the reactivation from latency. Although HIV can be reactivated by treatments that induce P-TEFb, the role of P-TEFb in Tat-related latency remains to be established. Using the latently infected cell lines generated in aim 1, we will examine the correlation between viral production and P-TEFb induction following exposure to cytokines and pharmacological agents that are known to activate T cells and macrophages. We will use RNA interference and inhibitors to determine directly whether P-TEFb is implicated. 3. Characterize the regulation of Tat and cyclin T1 by the cellular factors HIC and granulin. The ability of P-TEFb to support Tat transactivation can be positively or negatively modulated by cellular proteins that interact with cyclin T1 and Tat. We have isolated two such proteins, namely HIC (human I-mfa domain containing protein) and granulin, which bind to both Tat and cyclin T1 and regulate transactivation of the HIV-1 promoter. We will explore the ability of HIC and granulin to regulate transactivation of the HIV promoter and define the sequences of these proteins necessary for binding to cyclin T1 and Tat.